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BACKGROUND: Knowing that adenoviral vectors could initiate innate immunity, the ability of E1-deleted recombinant adenovirus (Ad-E1Delta) in induction of B7.1 and IL-2 molecules was studied. METHODS: The expression of green fluorescent protein in C1498 cells following transfection of these cells with adenovirus green fluorescent protein vector confirmed the ability of adenovirus vectors in infecting the cells and inducing the expression of the gene of interest. The expression of B7.1 molecule on the surface of the cells was assayed upon infection with Ad-E1Delta vector. Adenovirus-IL-2/B7.1 vector capable of inducing IL-2 and B7.1 expression in the cells was used as the positive control vector. RESULTS: According to the FACS results, about 4.17% of normal cells expressed B7.1 on their surface, while this level was increased in Ad-E1Delta transduced cells up to 14.43%. These results demonstrate that Ad-E1Delta vector considerably (about 3 folds) increases the expression of B7.1 on the cells. No detectable IL-2 was secreted into the medium of non-transduced and Ad-E1Delta transduced cells. CONCLUSION: Data indicate that the infection of C1498 cells with recombinant adenoviruses stimulates expression of B7.1 on the cell surface rather than secretion of IL-2 into the medium. 相似文献
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Sadigh ZA Shahrabadi MS Shafyi A Bambai B 《Pakistan journal of biological sciences: PJBS》2008,11(9):1220-1226
In this study, one of the measles virus membrane proteins, named hemagglutinin (H) which has a key role in tropism, receptor binding, hemagglutinating activity and also induction of protective immunity against viral infection, was expressed by the baculovirus expression system using specific plasmid (pDONR221) to produce entry clone. Measles Virus (AIK-C strain) genome was extracted from infected Vero cells. H gene was amplified by specific primers during RT-PCR reaction and inserted into the specific plasmid (pDONR221) using BP recombination reaction. Recombinant baculovirus harboring H gene was consequently constructed by LR reaction. Insect cells (Sf9) were infected with recombinant baculovirus. In order to increase viral titer, recombinant baculoviruses were passaged four times in Sf9 cells. Synthesis of H protein was verified by SDS-PAGE, western-blot and indirect immunoflourescene using goat polyclonal antibody against Measles Virus. The results showed that H protein was partially glycosylated, but it appeared to be active in hemagglutination assay. 相似文献
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侵染性克隆是研究病毒的重要工具,而偏大的病毒基因组及其在大肠杆菌中的不稳定给构建侵染性克隆造成很大困难。通常采用插入内含子和酵母同源重组等方式可以获得稳定克隆,但本实验最初利用酵母同源重组系统并未成功构建番木瓜畸形花叶病毒(PLDMV)的侵染性克隆。经研究证实,该不稳定现象确实存在于大肠杆菌中,而非酵母和农杆菌细胞。通过改良酵母同源重组方法,成功构建了有/无内含子intron 2的PLDMV侵染性克隆p35S-FL和p35S-FL-In2,农杆菌注射接种番木瓜均发病,侵染效率达64.7%~69.7%。本研究通过酵母同源重组质粒直接转化农杆菌,建立了一种10 d内即可稳定快速构建PLDMV侵染性克隆的E.coli-Free酵母同源重组新方法。该方法对其他在大肠杆菌中不稳定的植物病毒侵染性克隆的构建具有重要意义。 相似文献
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通过PCR方法,从E.coliJM109基因组DNA中扩增到全长为1296bp的glgC基因,对其进行定点突变,获得第296位和336位氨基酸同时突变的glgC336基因。将2者亚克隆进pET-30a,成功构建了重组表达载体pET-C和pET-C336,转化大肠杆菌BL21(DE3),在1mmol/LIPTG诱导下进行表达。SDS-PAGE电泳分析显示,在约53kD处有1条明显的蛋白表达带,证明目的基因已得到高效表达,且重组蛋白表达量占菌体蛋白总量的77.3%。 相似文献
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类花生致敏原3(iso-ARah3)是花生致敏原3的同系物,其表达活性与干旱及黄曲霉侵染相关。本研究通过RT-PCR技术从花生种子cDNA文库中克隆获得iso-ARah3的开放阅读框(ORF),全长1533bp,编码510个氨基酸,N端预测有20个氨基酸的信号肽序列。通过序列比对发现,该克隆序列有1处缺失突变,其N端210个氨基酸序列与胰蛋白酶抑制因子的同源性较高,达83.60%。通过构建原核表达载体pET28a-isoARah3,转化进大肠杆菌BL21,经IPTG诱导表达,利用SDS-PAGE检测表明,融合蛋白His-isoARah3获得高效表达,分子量约54kD。His-isoARah3经纯化和富集,对新西兰兔进行4次免疫,纯化获得的iso-ARah3多克隆抗血清,通过间接ELISA检测,表明获得了效价比(1∶512000)很好的抗体。通过对融合蛋白的诱导前和纯化后样品进行Western Blot分析,结果显示仅在纯化样品相应位置(54kD)有明显信号,表明所制备的抗体具有很高灵敏度和特异性。本研究结果为深入研究花生iso-ARah3基因的功能奠定了基础。 相似文献
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Maryam Ahmadi Narges Damavandi Mohammad Reza Akbari Fatemeh Davami 《Iranian Biomedical Journal》2016,20(2):68-76
Mammalian expression systems, due to their capacity in post-translational modification, are preferred systems for biopharmaceutical protein production. Several recombinant protein systems have been introduced to the market, most of which are under clinical development. In spite of significant improvements such as cell line engineering, introducing novel expression methods, gene silencing and process development, expression level is unpredictable and unstable because of the random location of integration in the genome. Site-specific recombination techniques are capable of producing stable and high producer clonal cells; therefore, they are gaining more importance in the biopharmaceutical production. Site-specific recombination methods increase the recombinant protein production by specifically inserting a vector at a locus with specific expression trait. The present review focused on the latest developments in site-specific recombination techniques, their specific features and comparisons. 相似文献
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Mirmomeni MH Kiani S Sisakhtnezhad S 《Pakistan journal of biological sciences: PJBS》2008,11(11):1497-1501
This study is directed towards the method of amplifying and cloning the SopE gene, that encodes Salmonella outer protein E. Strains used in this study were S. dublin collected from Kermanshah province. Genomic DNA was extracted by the general boiling method. Using the specific primers, a part of SopE gene was multiplied. The PCR product was inserted into the cloning vector (pTZ57R/T). Furthermore, E. coli DH5alpha bacteria were transformed to amplify the recombinant plasmid. Recombinant clones were identified by blue/white selection. Recombinant plasmids were purified by alkaline lysis procedure. Moreover, identity of the SopE/pTZ57R/T product was confirmed by restriction enzyme digestion assay and sequencing. Finally, the cloned gene was compared with that published by the NCBI Genbank (L78932). The results showed that the obtained sequence differed in four nucleotides which resulted in two amino acid differences. The cloned SopE was submitted to the NCBI Genbank (EU399750). 相似文献
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利用PCR方法克隆香蕉束顶病毒海南分离物(Banana bunchy top virus,BBTV-HN)DNA2编码区,将其插入到原核表达载体pGEX-6p-1谷胱苷肽-S-转移酶(GST)基因下游.所得克隆pGEX-HN-B2测序结果表明,插入的DNA2片段为267 bp,且表达相位和理论上设计的完全一致.把此重组质粒转化到E.coli Rosetta(DE3)中,经过异丙硫代半乳糖苷(IPTG)诱导后出现约33 Ku融合蛋白质表达条带.为获得特异的血清,切下目的表达条带并免疫兔子.经Western-blotting检测,获取的血清可以和表达的33 Ku融合蛋白特异结合,表明已成功获得DNA2编码蛋白的血清. 相似文献
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应用PCR方法从感染香蕉束顶病毒的香蕉植株幼嫩假茎和叶片总DNA中克隆了Rb(Rb-binding-like protein)基因的编码区,将其插入原核表达载体pET-28b中构建重组质粒pET28b-Rb。转化重组质粒的E.coli BL21(DE3)进行IPTG诱导表达,SDS-PAGE分析结果表明,表达产物大小为22.6ku,且主要以包涵体形式稳定表达。目的蛋白经Ni2+-NTA琼脂糖亲和层析纯化后免疫家兔并获得抗血清。Western blot检测结果表明,抗血清与诱导表达的BBTV编码Rb蛋白发生特异性反应。间接ELISA法测定的抗血清效价大于1:250000。用抗血清对感病香蕉和健康香蕉进行检测,结果表明,抗血清对感病香蕉有很高特异性,最佳工作浓度为1:1500。 相似文献
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抗菌肽B基因的点突变及在昆虫细胞中的表达 总被引：9，自引：0，他引：9
抗菌肽Ｂ基因经ＰＣＲ定点突变技术改造后,克隆到杆状病毒转移载体ｐＡｃＧＰ６７Ｂ中的信号肽位点上,随后与野生ＡｃＮＰＶ－ＤＮＡ共转染Ｓｆ９细胞,经空斑筛选、ＰＣＲ检测证实获得含该基因的重组病毒。重组病毒在Ｓｆ９细胞中扩增并表达抗菌肽Ｂ基因。取含表达产物的细胞培养液检测其生物活性表明：改造过的抗菌肽Ｂ基因在Ｓｆ９细胞中获得初步表达,且表达的分泌型产物对ＥｓｃｈｅｒｉｃｈｉａｃｏｌｉＫ１２Ｄ３１有抑菌活性。 相似文献
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本研究克隆鉴定了香蕉枯萎病菌4号生理小种（Foc4）的细胞周期蛋白C1（Fusarium Cyclin C1,FCC1）基因FocFCC1,采用Split-marker同源重组技术获得基因敲除突变体ΔFocFCC1,通过比较突变体和野生菌株在生长速度、产孢量和致病力等方面的差异,探索了FocFCC1基因缺失对香蕉枯萎病菌生物学功能的影响,并评估了FocFCC1作为枯萎菌内源报告基因的可行性。结果表明：与Foc4野生型菌株相比,ΔFocFCC1菌丝生长缓慢、菌丝畸形及产孢量减少,对巴西蕉苗的致病力明显减弱,推测FocFCC1基因在Foc4的生长发育、产孢及致病力等方面具有重要作用。以FocFCC1为靶标基因,评估了两种基因敲除重组方法介导的基因敲除效率,利用红色表型作为判断依据,挑取再生板上有红色表型的转化子进行PCR检测,Split-marker重组方法获得阳性转化子比例为91.7%,而多片段装配融合方法获得阳性转化子的比例为64%。ΔFocFCC1出现典型红色菌落,红色表型与PCR检测的FocFCC1阳性敲除转化子一一对应,FocFCC1基因敲除后红色表型肉眼容易分辨,可作为香蕉枯萎病菌内源报告基因探索新的遗传操作技术在该菌上应用的可行性。 相似文献
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水稻稻曲病菌G蛋白β亚基基因的克隆、表达与序列分析 总被引：2，自引：0，他引：2
水稻稻曲病是由稻绿核真菌[Ustilaginoidea virens(Cooke)Tak]引起的一种常见的水稻后期穂部病害。G蛋白可能参与了稻曲病菌的致病过程。为了研究G蛋白在病菌致病过程中的作用,分离并分析了稻曲菌的G蛋白β亚基编码基因。根据丝状真菌G蛋白β亚基编码基因的同源保守序列设计简并引物,采用同源克隆和热不对称交错PCR的方法,分离得到了稻曲菌的G蛋白β亚基全编码基因序列。该序列的长度为2037bp,包含4个内含子,5个外显子和1个编码359个氨基酸的开放阅读框。根据克隆到的UvGβ1设计引物,通过RT-PCR克隆到包含整个开放阅读框的cDNA序列。该基因的DNA序列和cDNA序列在GenBank中注册的登记号分别为GU014921和GU065745。系统进化分析表明该片段与栗疫病菌(Cryphonectria parasitica)的G蛋白β亚基基因亲缘关系最近。将该基因的整个开放阅读框连接于pET-30a构建原核表达载体,通过诱导获得了重组蛋白。 相似文献
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一个水稻小热休克蛋白的异源表达及寡聚特性分析 总被引：1，自引：1，他引：0
【目的】在前期研究中,本实验室已克隆了一个水稻小热休克蛋白基因(OsSHSP17.6),并发现该基因的表达明显受到热激和病毒侵染调控,表明该蛋白可能在逆境胁迫过程中起重要作用。本研究的目的在于进一步明确OsSHSP17.6的特性。【方法】在本研究中,进一步将该基因亚克隆至原核表达载体p ET-32a并导入大肠杆菌E.coli BL21(DE3)pLysS诱导表达,通过亲和层析的方法纯化了该重组蛋白,进一步用于非变性聚丙烯酰胺凝胶电泳和Western blotting分析。【结果】异源表达的重组OsSHSP17.6能减轻IPTG对宿主菌的毒害。非变性聚丙烯酰胺凝胶电泳和Western blotting分析显示纯化的重组OsSHSP17.6蛋白在体外能形成同源二聚体和寡聚体。【结论】这些结果支持OsSHSP17.6是一个有功能的分子伴侣蛋白并表明该蛋白可能通过形成同源寡聚体的方式参与逆境胁迫反应,这为进一步明确OsSHSP17.6的功能机制奠定基础。 相似文献
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短短芽胞杆菌FJAT-0809-GLX具有抑菌、抗褐变和保鲜功能。以短短芽胞杆菌菌株FJAT-0809-GLX总DNA为模板,采用PCR扩增超氧化物歧化酶(Supseroxide dismutase,SOD)基因,将该片段纯化回收后与p MD18-T连接并转入大肠杆菌(Escherichia coli)DH5α,进行序列测定,结果显示SOD基因序列长度为609 bp(Gen Bank登录号:KM255665),编码202个氨基酸残基。将SOD基因片段与同样酶切的表达载体p ET-28a连接,构建重组表达载体p ET-28a-SOD,转入大肠杆菌BL-21,采用异丙基-β-D-硫代吡喃半乳糖苷(Isopropyl-β-D-thiogalactopyranoside,IPTG)进行诱导表达。SDS-PAGE结果表明,在菌体中存在约25 ku的蛋白表达产物,与该基因ORF预期大小接近。以上结果为进一步研究该酶的生理生化特性奠定了基础。 相似文献